Legacy effects in temporally separated tadpole species are not mediated by invasive Western Mosquitofish (Gambusia affinis)

Abstract Temporally separated species are often thought to have limited competition over a shared resource. However, early arriving species may consume a limited resource such that later‐arriving species have access to fewer resources and thus experience competitive effects, even if they are temporally separated (i.e., they experience legacy effects from the early species). The presence of a predator might affect potential legacy effects by influencing the behavior or survivorship of the early species. Using a mesocosm experiment, I examined whether the presence of nonnative Western Mosquitofish (Gambusia affinis) mediated legacy effects in the interaction of two temporally separated species of tadpoles, early arriving American Toads (Anaxyrus americanus) and late‐arriving Bullfrogs (Rana catesbeiana). Anaxyrus americanus tadpoles reduced R. catesbeiana tadpole growth despite all A. americanus tadpoles metamorphosing 8 days before the introduction of R. catesbeiana tadpoles into the mesocosms (i.e., legacy effects). Gambusia affinis had limited effects on A. americanus (1 day delay in metamorphosis but no effect on survivorship or size at metamorphosis) and positive effects on R. catesbeiana (increased growth). There were no significant interactions between the A. americanus tadpole density and G. affinis treatments. In conclusion, I found evidence of significant legacy effects of A. americanus tadpoles on R. catesbeiana tadpoles, but no evidence that G. affinis mediated the legacy effects.


| INTRODUC TI ON
The timing of arrival of one species relative to the arrival of a potential competitor (i.e., earlier, at the same time, or later) can determine the extent of competition between the species (e.g., Blackford et al., 2020). Temporally separated species or those that have minimal seasonal overlap may have reduced or eliminated competition over a shared resource (Lawler & Morin, 1993;Wilbur, 1980), as has been shown in a variety of taxa (McKane et al., 1990;Monceau et al., 2015;Sladecek et al., 2017). However, some temporally separated species have been shown to compete (e.g., Barnes & Murphy, 2023;Branson, 2010;Faeth, 1986;Kaplan & Denno, 2007), presumably via legacy effects which occur when the effects of a species are evident in a community even after the species is no longer present (Cuddington, 2011).
The breeding phenology of anurans in ponds frequently leads to tadpoles of different species occupying the same pond but not overlapping, or only partially overlapping, temporally. The presence of legacy effects of an early species affecting a later species in anuran larvae is therefore potentially important. Legacy effects have been demonstrated in larval anuran communities. For example, Hyla andersonii tadpoles introduced into mesocosms later in the season grew and developed slower than those introduced earlier in the season, consistent with the later season tadpoles experiencing lower resources due to consumption of resources (e.g., periphyton) by early season conspecific and Anaxyrus woodhousii tadpoles (Morin et al., 1990). The presence of Anaxyrus americanus in the spring lowered the survivorship and delayed metamorphosis of Hyla chrysoscelis tadpoles even though they did not overlap temporally, likely because of shifts they caused in the composition of the algal community resulting in an increase in the prevalence of lower-quality algal species (Wilbur & Alford, 1985). Scaphiopus holbrooki tadpoles had a negative competitive effect on the growth of Hyla chrysoscelis despite no temporal overlap; however, Anaxyrus americanus did not (Wilbur, 1987). The presence of Hyla crucifer tadpoles reduced the survival and mass of metamorphosis of H. versicolor even though the H. crucifer was almost all metamorphosed before the H. versicolor were added (Morin, 1987). Thus, early arriving larval anuran species may be able to competitively affect later-arriving larval anuran species through a shared resource, such as periphyton or algae, and thus reduce the growth or survivorship of the later species, even if there is no, or very limited, temporal overlap.
The importance of competitive legacy effects in communities could be mediated by predators. Previous work has demonstrated that the outcome of competition among simultaneously occurring anuran tadpole species can be altered by the presence of a predator. For example, the presence of a predator may allow competitively weaker species of tadpoles to persist in experimental communities with competitively superior species because they differentially prey on the superior competitive species or they cause a reduction in the overall density of tadpoles limiting the impact of competition (Morin, 1981(Morin, , 1983Relyea & Rosenberger, 2018;Rudolf & Roman, 2018;Smith, 2006). Second, the predator can induce a behavioral response such that the impact of the first species on a shared resource is reduced, such as through inhibition of foraging activity or through changes in morphology related to consumption (e.g., mouth parts) between the two species (Relyea, 2000;Werner, 1991;Werner & Anholt, 1996). Thus similar mediation of competitive legacy effects could arise if the early arriving species is more strongly affected by the predator than the late-arriving species. Indeed, the presence of a predator reduced the competitive effects of an early arriving species (Pseudacris crucifer) on a late-arriving species (Hyla versicolor) that temporally overlapped briefly as tadpoles (Morin, 1987).
One potential predator that could influence competitive legacy effects in anuran larval communities is the Western Mosquitofish (Gambusia affinis). Gambusia affinis is among the most widely introduced and successful invasive freshwater fishes (Bernery et al., 2022). Mosquitofish (Gambusia affinis and G. holbrooki) have been introduced into a variety of freshwater ecosystems around the world and can have dramatic negative effects, such as population declines or extinctions, on invaded aquatic communities, including amphibians (Pyke, 2008). Gambusia can also have negative effects on amphibians in their native range (Baber & Babbitt, 2003).
However, the effects of fish can vary among species of amphibians based on their susceptibility to fish predation. For example, the tadpoles of the relatively unpalatable Bullfrog (Rana catesbeiana) can benefit from the presence of fish through the consumption of potential competitors (e.g., Werner & McPeek, 1994) or invertebrate predators (e.g., Smith et al., 1999;Werner & McPeek, 1994). Thus, the consequences of the presence of a fish predator, especially an introduced species such as G. affinis, on anuran larval communities can be complex.

I examined whether the presence of nonnative Western
Mosquitofish (Gambusia affinis) mediates legacy effects in the interaction of two temporally separated species of tadpoles, early arriving American Toads (Anaxyrus americanus) and latearriving Bullfrogs (Rana catesbeiana). There is evidence that A. americanus and R. catesbeiana tadpoles are potential competitors over a shared algal resource (e.g., Boone et al., 2007;Seale & Beckvar, 1980). Previous studies using A. americanus and R. catesbeiana from the same source populations as those used in my experiment examined the effects of G. affinis on the survivorship, growth, and behavior of their tadpoles. Local A. americanus tadpoles did not alter activity in the presence of G. affinis (Smith et al., 2008(Smith et al., , 2009). However, G. affinis had a negative effect on the survivorship and size at metamorphosis of A. americanus in a mesocosm experiment (Smith & Dibble, 2012), as did Bluegill (Lepomis macrochirus) , suggesting they are susceptible to fish predators. Local R. catesbeiana did not alter their activity in the presence of G. affinis in laboratory experiments (Smith et al., 2008(Smith et al., , 2009  tadpoles of R. catesbeiana (mean mass = 0.040 ± 0.002 g) to each mesocosm on 27 June, after all American Toads had metamorphosed. Tadpole densities were at the low end of naturally occurring densities of tadpoles of these and other species observed in local ponds (e.g., Smith et al., 2003Smith et al., , 2005. The density of G. affinis used was also within the range of densities of G. affinis found in local ponds (range = 1-32 m −2 ; J. E. Rettig and G. R. Smith, unpublished data). In addition, I have used similar numbers of G. affinis in other mesocosm experiments that had high survivorship of G.
Water levels in mesocosms were allowed to fluctuate with evaporation and precipitation.
For A. americanus, I allowed metamorphosis to occur, removing metamorphs daily when both forelimbs had emerged. To describe the time to metamorphosis, I used the median days to metamorphosis for each mesocosm rather than the mean because the median is less affected by any outlier individuals that would have an outsized impact on the mean. Metamorphs were housed in the laboratory in plastic containers with access to water until the tail was resorbed. I then measured their SVL to the nearest 0.01 cm with digital calipers and weighed them to the nearest 0.001 g using an electronic balance.
In central Ohio, tadpoles of R. catesbeiana overwinter, and therefore, no R. catesbeiana metamorphosed during the experiment. At the end of the experiment (18 July), I removed all surviving R. catesbeiana tadpoles from mesocosms and weighed them to the nearest 0.001 g using an electronic balance (after blotting dry with a paper towel). I also removed G. affinis from the mesocosms and counted all individuals. Reproduction of G. affinis occurred in all mesocosms in which they had been stocked; however, the number of G. affinis at the end of the experiment did not differ among the A. americanus treatments (F 2,12 = 2.05, p = .17).
I collected water from each mesocosm on 5 dates throughout the experiment (25 May, 3 June, 10 June, 20 June, and 11 July) and estimated total chlorophyll a (chl a) levels using standard fluorometric methods (Welschmeyer, 1994). Periphyton levels at the end of the experiment were estimated by removing periphyton from a predetermined area of the south-facing internal mesocosm wall In mesocosms with G. affinis, the median number of days to metamorphosis was more than a full day later than in mesocosms without G. affinis (Figure 1

| Rana catesbeiana
Gambusia affinis had no effect on R. catesbeiana tadpole survivorship

| Primary producers
Mesocosms without G. affinis tended to have more filamentous algae than mesocosms with G. affinis, however, this trend was not statistically significant at the α = 0.05 level (Figure 3b There was less chl a in mesocosms with no G. affinis than in mesocosms with G. affinis (Figure 4a; F 1,24 = 15.6, p = .0006). Overall, there was no effect of A. americanus tadpole density (F 2,24 = 1.60,  The three-way interaction between time, G. affinis treatment, and A. americanus tadpole density treatment was not significant (F 10,40 = 1.87, p = .079).

| DISCUSS ION
In the simple community studied in my experiment, A. americanus tadpoles had a significant negative effect on R. catesbeiana tadpoles despite the fact that the vast majority of A. americanus tadpoles had metamorphosed 2-3 weeks, and all had metamorphosed by 8 days,

F I G U R E 2
The effects of the interaction between the presence (closed circles) and absence (open circles) of Gambusia affinis and initial Anaxyrus americanus tadpole density on the (a) survivorship, (b) mean body mass, and (c) total biomass of Rana catesbeiana tadpoles. * Indicates a significant effect of G. affinis treatment. Shared letters indicate means that are not significantly different. Means are given ±1SE.

F I G U R E 3
The effects of the interaction between the presence (closed circles) and absence (open circles) of Gambusia affinis and initial Anaxyrus americanus tadpole density on (a) amount of filamentous algae and (b) mean periphyton mass. Means are given ±1SE.
before the introduction of R. catesbeiana tadpoles into the mesocosms (i.e., there were significant legacy effects). Anaxyrus americanus tadpoles negatively affected the mean individual mass and total biomass of R. catesbeiana tadpoles at the end of the experiment, but did not affect their survivorship. The negative effects of the early arriving A. americanus tadpoles on later-arriving R. catesbeiana tadpoles that we observed are consistent with the competitive effects of A. americanus on later-arriving tadpoles in other studies (Distel & Boone, 2011), including when they did not overlap (Wilbur & Alford, 1985). However, other experiments have demonstrated that the consumption of algal resources by early tadpoles can lead to reduced availability of those resources to later tadpoles (e.g., Hernandez & Chalcraft, 2012;Morin et al., 1990;Rowland et al., 2017). In addition, I have found reduced periphyton levels in mesocosms with tadpoles, as well as a reduction in periphyton with higher densities of tadpoles, in other similar experiments Smith & Burgett, 2012;G. R. Smith, M. Smyk, M. Jones, and J. Hollis, unpublished data). Wilbur and Alford (1985) also suggested that persistent effects of A. americanus on later tadpoles were likely due to an increase in the prevalence of lower-quality algae species as a result of consumption by the A. americanus tadpoles.
Similarly, overwintered Rana sphenocephalus tadpoles had a negative effect on Bufo terrestris tadpoles, probably due to the reduction of algal resources by the overwintered R. sphenocephalus tadpoles prior to the introduction of the B. terrestris tadpoles (Hernandez & Chalcraft, 2012). It is also possible that A. americanus had some other effects on the mesocosm environments in which they occurred. For example, there is some evidence that tadpoles can release growthinhibiting factors or otherwise condition the water in such a way that reduces the growth or survivorship of competitors (Beebee, 1995;Griffiths, 1995;Steinwascher, 1981; but see Cabrera-Guzmán et al., 2013;Petranka, 1989Petranka, , 1995. In my experimental amphibian larval community, G. affinis caused a delay of slightly more than one day in the median number of days to metamorphosis in A. americanus, but had no effect on the proportion of tadpoles reaching metamorphosis or on size at metamorphosis. For R. catesbeiana, the presence of mosquitofish increased the mean mass of the tadpoles, as well as the total biomass of tadpoles produced. The presence of fish can benefit R. catesbeiana by the consumption of invertebrate predators (e.g., Smith et al., 1999;Werner & McPeek, 1994). Gambusia affinis does indeed reduce the number of invertebrate predatory species, such as odonates and dytiscid beetle larvae colonizing experimental ponds in central Ohio (Harmon & Smith, 2021). However, this effect is unlikely in our experiment since colonization of mesocosms by macroinvertebrates was prevented. Instead, I postulate that effects on primary productivity may be responsible for the positive effect of G. affinis on R. catesbeiana. The presence of G. affinis had significant effects on primary productivity at the end of this experiment, with more chl a and a tendency for less filamentous algae. Predators can indirectly positively affect prey by making nutrients more available to primary producers thus potentially increasing the resources available to the tadpoles (e.g., Costa & Vonesh, 2013). For example, G. affinis can affect nutrient recycling through excretion and influence primary productivity (Akhurst et al., 2012;Hargrave, 2006;Hurlbert et al., 1972). Indeed, the presence of G. affinis or G. holbrooki can increase periphyton and/ or phytoplankton (Akhurst et al., 2012;Fryxell et al., 2015;Smith et al., 2013). Gambusia affinis can also increase primary productivity (chl a, periphyton) by removing primary consumers, such as zooplankton in similar mesocosm experiments (Rettig & Smith, 2021).
However, the presence of G. affinis may not always have cascading effects on phytoplankton or periphyton (e.g., Geyer et al., 2016;Smith & Dibble, 2012). Thus, there appear to be several, not mutually exclusive, explanations of the positive effects of G. affinis on R. catesbeiana in this experiment.
Based on other studies that found negative effects of G. affinis on A. americanus (Smith & Dibble, 2012)

CO N FLI C T O F I NTE R E S T S TATE M E NT
The author declares that he has no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data underlying this manuscript are archived on Dryad at https://doi.org/10.5061/dryad.573n5 tbcg.